High-performance liquid chromatographic determination of oxolinic acid and flumequine in the live fish feed Artemia

A high-performance liquid chromatography (HPLC) analytical method for the determination of oxolinic acid and flumequine in Artemia nauplii is described. The samples were extracted and cleaned up by a solid-phase extraction (SPE) procedure using SPE C₁₈ cartridges. Oxolinic acid and flumequine were determined by reversed-phase HPLC using a mobile phase of methanol–0.1 M phosphate buffer, pH 3 (45:55, v/v) and a UV detection wavelength of 254 nm. Calibration curves were linear for oxolinic acid in the range of 0.2–50 μg/g (r²=0.9998) and for flumequine in the range of 0.3–50 μg/g (r²=0.9994). Mean recoveries amounted to 100.8% and 98.4% for oxolinic acid and flumequine, respectively. The quantification limit was 0.2 μg/g for oxolinic acid and 0.3 μg/g for flumequine. Quantitative data from an in vivo feeding study indicated excellent uptake of both drugs by Artemia nauplii.     

Changes in biomass, lipid, fatty acid and elemental composition during the abbreviated larval development of the subantarctic shrimp Campylonotus vagans

Ontogenetic changes in biomass and chemical composition were studied in the laboratory during the abbreviated larval and early juvenile development of the caridean shrimp Campylonotus vagans from the subantarctic Beagle Channel, Argentina. At 7±0.5 °C, development from hatching to metamorphosis took about 44 days. The larvae started feeding on Artemia nauplii immediately after hatching, although larval resistance to starvation was high (average 18 days, maximum 29 days). Dry mass (DM), carbon (C), nitrogen (N) and hydrogen (H) contents increased about a fourfold from hatching to metamorphosis, while the C:N mass ratio increased from about 3.7 to 4.3. The protein and total lipid contents increased gradually from hatching to the first juvenile stage, the former from 190 to 640 μg/individual, the latter from 37 to 95 μg/individual. The lipid mass fraction was low throughout larval development (3-9% of DM), while the protein content was much higher and almost constant (30-40%). The dominating fatty acids were 18:1(n-9), 16:0, 20:5(n-3), 18:1(n-7), 18:3(n-3), 18:0, 16:1(n-7). Except for 20:5(n-3), these resulted mainly from food uptake (Artemia nauplii). Exuvial losses of C, H and N (all larval stages combined) accounted for only 7%, 1% and 1% of the initial values at hatching. In contrast, 37% of initial DM was lost. Partially food-independent (endotrophic) larval development is discussed as an adaptation to food scarcity at high latitudes, while the abbreviated planktotrophic larval development appears to be synchronised with seasonal peaks in primary production, allowing for an optimal resource exploitation in a food-limited environment.     

New high-performance liquid chromatographic method for amphotericin B analysis using an internal standard

A simple and reproducible HPLC method for the analysis of amphotericin B (AmB) in serum, lung and liver using natamycin as the internal standard was developed. AmB and natamycin were extracted from serum, lung and liver and were separated using an isocratic elution from a C₁₈ reversed-phase column. The mobile phase consisted of acetonitrile-10 mM acetate buffer pH 4.0 (37:63, v/v). The HPLC system had two detectors in series. One was set at 303 nm and the other at 383 nm for the detection of natamycin and AmB, respectively. The retention times of AmB and natamycin were 15 and 6 min, respectively. The recovery efficiency was 96-70%. The limit of quantification was 0.1 μg/ml. The assay was reproducible, the within-day coefficient of variation (n=6) was <8% for serum, lungs and liver. The between-day variability (n=6) was <7.7% for serum, liver and lungs at 1 μg/ml or 1 μg/g tissue concentration. The assay was linear within the range 1–40 μg/ml (r²=0.999).     

Ion-pair high-performance liquid chromatographic assay method for the assessment of clarithromycin stability in aqueous solution and in gastric juice

A simple and selective ion-pair HPLC method has been developed for the analysis of clarithromycin in aqueous solutions and in gastric juice. A Hypersil ODS 5-μm (150 × 4.6 mm I.D.) column was used with a mobile phase consisting of acetonitrile-aqueous 0.05 M phosphate buffer (pH 4.6) containing 5 mM 1-octanesulphonic acid (50:50, v/v). The column temperature was 50°C and detection was by UV absorption (210 nm). The limits of detection of 50-μl samples were 0.4 μg/ml (aqueous) and 0.78 μg/ml (0.5 ml gastric juice) or better. The assay was linear in the range of 1.56 to 100 μg/ml with r² values greater than 0.99. The recovery from the gastric juice samples was 98.5±2.9%. The method was applied successfully to determine the stability of clarithromycin in 0.01 M HCl and gastric juice.     

Simultaneous determination of sulphamonomethoxine and its N4-acetyl metabolite in blood serum by high-performance liquid chromatography with direct injection

A high-performance liquid chromatographic method with direct injection has been developed for the simultaneous determination of sulphamonomethoxine and its N⁴-acetyl metabolite in serum of animals and fish. A HISEP shielded hydrophobic-phase column (15 cm × 4.6 mm I.D.), a mobile phase of 0.05 M citric acid–0.2 M disodium hydrogenphosphate-acetonitrile (70:15:15, v/v), and ultraviolet detection at 265 nm were used. The standard calibration curves in serum of chicken, pig, cattle, rainbow trout and yellowtail were linear over the range 0.5–20 μg/ml. The recoveries of sulphamonomethoxine and its N⁴-acetyl metabolite from all serum samples determined at different concentrations (0.5, 2.0 and 10.0 μg/ml) were 93–103% and 90–103%, respectively. The lowest measurable sulphamonomethoxine and N²-acetyl metabolite concentrations were 0.04 and 0.1 μg/ml, respectively, for all serum samples.     

An Efficient and Reliable Method of DNA Extraction from Meyna spinosa — A Traditional Medicinal Plant from North-East India

A simple, efficient and reliable CTAB method is standardized for genomic DNA isolation from fresh young leaves of a traditional medicinal plant Meyna spinosa. Key steps in the modified procedure include additional chloroform: isoamyl alcohol (24:1, v/v) extraction, addition of 4% PVP in the extraction buffer and an overnight isopropanol precipitation at room temperature. This procedure yields a high amount (46 μg DNA g^?1 fresh leaf tissue) of good quality DNA free from contaminants. The isolated DNA is suitable for digestion with EcoRI and HindIII restriction enzymes and can be used in other DNA manipulation techniques.     

Determination of piromidic acid residues in trout muscle tissue and in urine by liquid chromatography with post-column modification of pH and fluorimetric detection

A rapid high-performance liquid chromatographic method has been developed to determine piromidic acid in trout muscle tissue and in urine, in the presence of nalidixic, 7-hydroxymethylnalidixic, oxolinic and pipemidic acids and cinoxacin. A Nova-Pak C₁₈ column was used with acetonitrile–4·10⁻⁴ M oxalic acid (40:60, v/v) as the mobile phase. A post-column change of pH was made with NaOH. Fluorimetric detection at 456 nm (λ_ex 275 nm) was used. The instrumental detection limit was 5.91 ng/ml, based on height of peak. Pretreatment of the urine samples was not necessary and fish samples were extracted with sodium hydroxide solutions and cleaned by means of an extraction with chloroform. Detection limit was 147 ng/ml for urine and 5.91 ng/g for trout muscle. Good separation without interference from any other components was obtained. Recovery was better than 87% in urine and better than 72% in trout muscle tissue.     

Filter feeding in the hermit crab

Summary The hermit crab, Pagurus bernhardus is able to remove both Artemia nauplii and unicellular algae from suspension. Crabs with wet weights of 1.1–9.2 g consumed all of the 300 Artemia nauplii contained in 200 ml of sea water within 1 h. Crabs weighing 0.7–1.1 g wet weight filtered suspended Dunaliella algae (8 m) from concentrations of 10–350 million cells per liter at a rate of 26% and 47% within 2 and 5 h, respectively. A similar result was obtained with an 11 g crab. During filter feeding activity a water current is generated by the flagella of the exopods of the second and third maxillipeds. Artemia nauplii are caught by grasping movements of the endopods of the third maxillipeds, whereas filtering of unicellular algae is probably achieved by the two maxillae. It is assumed that filter feeding activity depends on the same structures and behavior as in deposit feeding. P. bernhardus is one more example of a benthic marine animal which may use any food source which becomes available in the course of the seasons.     

Feeding rates of planktonic copepods from a tropical sea

The feeding rates of the marine planktonic copepods, Eucalanus subcrassus Giesbrecht, Tortanus gracilis (Brady), Calanopia elliptica (Dana) (both male and female), Temora turbinata (Dana), and Paracalanus aculeatus Giesbrecht (only female) from tropical inshore waters have been studied. Newly hatched Artemia nauplii (for Eucalanus, Tortanus, and Calanopia), Dunaliella (for Temora), and Skeletonema (for Paracalanus) were used as food.Feeding rates were measured for a single individual through successive incubations once or twice a day until death to determine changes in feeding rate after collection. Copepods survived from a few days to three weeks. In general, feeding rates varied from day to day, but were less variable than the differences between day and night rates. In some cases, feeding rate consistently decreased up to the death of the copepod. Daily ration, estimated in terms of percentage body weight, was in the range of 28–329 ‰ Using the results together with the those of other workers, gives the relation between daily ration (Y, % body weight) and body weight of copepods (X; μg dry weight) at 20 °C as, logY = 2.531?0.377 logX.Copepods given Artemia nauplii as food killed more nauplii than were eaten. This phenomenon, tentatively called ‘over-hunting’, is possibly an important feeding behaviour for carnivorous copepods.     

Response of bighead carp Aristichthys nobilis and Asian catfish Clarias macrocephalus larvae to free-living nematode Panagrellus redivivus as alternative feed

The use of Panagrellus redivivus as live feed for bighead carp and Asian catfish larvae was tested. In experiment 1, carp larvae were given Artemia nauplii (control) or Panagrellus twice daily for 21 days. A third treatment consisted of unfed larvae. The same three treatments were used in experiment 2 plus another with a commercial entomopathogenic nematode (EPN). Bighead carp larvae given Panagrellus in experiment 1 had much lower growth and survival than those fed Artemia nauplii. This could be due to low nematode density (5–30 mL^?1 water) during feeding. The unfed larvae had 100% mortality by days 11–13. In experiment 2, growth and survival of carp larvae given Artemia nauplii (5–10 mL^?1) and Panagrellus (50 mL^?1) did not differ significantly (P > 0.05). All unfed larvae had died by day 13, while larvae fed EPN were all dead by day 8. Two experiments on Asian catfish were likewise conducted. In experiment 1, the catfish larvae were fed Tubifex (ad libitum), Panagrellus (50–100 mL^?1 per feeding) orArtemia (5 nauplii mL^?1 per feeding) three times daily for 14 days. In experiment 2, larvae were fed Artemia alone (10 nauplii mL^?1 per feeding), Panagrellus alone (100 mL^?1 per feeding), or their combination with a 38% protein dry diet twice daily. For both experiments, catfish larvae fed Panagrellus had significantly lower growth and survival than those fed Tubifex or Artemia. The combination of Panagrellus and dry diet created little improvement in the growth and survival of catfish larvae.     

Stereoselective determination of R(−)- and S(+)-MK-571, a leukotriene D4 antagonist,in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic method that utilizes fluorescence detection was developed for the selective and sensitive quantification of R(−)- and S(+)-enantiomers of MK-571 (1), a potent and specific leukotriene D₄ antagonist, in human plasma. Racemic 1 was isolated from the acidified plasma using solid-phase extraction and the resulting residue was successfully reacted with isobutyl chloroformate and R(+)-1-(1-naphthyl)ethylamine in triethylamine—acetonitrile medium to form the diastereomer of each enantiomer. A structural analogue of 1 was used as internal standard. The derivatized sample was dissolved in 1,1,2-trichlorotrifluoroethane and an aliquot was chromatographed on a (R)-urea chiral column using a mobile phase containing 89% triethylamine—pentane (3:1000, v/v), 10% 2-propanol, and 1% acetonitrile at a flow-rate of 1.5 ml/min. The fluorescence response (excitation wavelength, 350 nm; emission wavelength, 410 nm) was linear (r²>0.999) for concentrations of enantiomers of 1 from 0.05 μg/ml, the lowest quantitation limit, up to 2.5 μg/ml. Intra-day coefficients of variation at 0.05 μg/ml were 2.4% for the R(−)-isomer and 2.0% for S(+)-isomer. The corresponding inter-day coefficients of variation for R(−)- and S(+)-1 were 2.6 and 3.6%, respectively. The utilit of the methodology was established by analysis of plasma samples from male volunteers receiving single intravenous and oral doses of racemic 1.     

High-performance liquid chromatographic determination of levodropropizine in human plasma with fluorometric detection

The present describes a new high-performance liquid chromatographic method with fluorescence detection for the analysis of levodropropizine [S-(−)-3-(4-phenylpiperazin-1-yl)-propane-1,2-diol] (Levotuss), an anti-tussive drug, in human serum and plasma. A reversed-phase separation of levodropropizine was coupled with detection of the native fluorescence of the molecule, using excitation and emission wavelengths of 240 nm and 350 nm respectively. The analytical column was packed with spherical 5 μm poly(styrene-divinylbenzene) particles and the mobile phase was 0.1 M NaH₂PO₄ pH 3-methanol (70:30, v/v), containing 0.5% (v/v) tetrahydrofuran. For quantitation, p-methoxylevodropropizine was used as the internal standard. Samples of 200 μl of either serum or plasma were mixed with 200 μl of 0.1 M Na₂HPO₄ pH 8.9 and extracted with 5 ml of chloroform-2-propanol (9:1, v/v). The dried residue from the organic extract was redissolved with distilled water and directly injected into the chromatograph. The limit of detection for levodropropizine, in biological matrix, was about 1–2 ng/ml, at a signal-to-noise ratio of 3. The linearity was satisfactory over a range of concentrations from 3 to 1000 ng/ml (r² = 0.99910); within-day precision tested in the range 5–100 ng/ml as well as day-to-day reproducibility proved acceptable, with relative standard deviations better than 1% in most cases. Interferences from as many as 91 therapeutic or illicit drugs were excluded.     

The effect of the nanoflagellateTetraselmis suecica on the growth and survival of grunion,Leuresthes tenuis,larvae

Synopsis The nanoflagellateTetraselmis suecica was tested both as the sole food source and as a diet complement toArtemia nauplii for grunion,Leuresthes tenuis, larvae. A total of 4800 grunion larvae, obtained through artificial insemination and incubation, were cultivated under laboratory conditions. Growth and survival rates were registered for 14 days in two experimental series. In the first series the nanoflagellateT. suecica was offered as the sole food source at five different concentrations. Survival and growth increased in agreement with the increase inT. suecica concentration. In the second series,Artemia nauplii were offered at six concentration levels. This series was divided into two groups: the nanoflagellateT. suecica was added to one group at a concentration of 5000 cells ml^–1; the other group was maintained without nanoflagellates. In this series, survival and growth were directly related to nauplii concentration, but significant effects of the nanoflagellates were evident only in relation to the survival; the greatest difference (58% without nanoflagellates vs. 69% with nanoflagellates) was observed at anArtemia concentration of 1000 nauplii 1^–1. The mechanism responsible for increased survival ofL. tenuis larvae in presence of phytoplankton is unclear.     

Fatty acid composition of Turbatrix aceti and its use in feeding regimes of Coregonus maraena (Bloch, 1779): is it really a suitable alternative to Artemia nauplii?

By incorporating the free‐swimming nematode Turbatrix aceti into early feeding regimes of the European whitefish Coregonus maraena, the suitability of this nematode species was investigated as an alternative to Artemia nauplii. During a 14‐day feeding trial in a total of 25 aquaria each 1.7 L (each treatment n = 5, 255 larvae/tank) T. aceti was used either as the sole live food or in combination with Artemia nauplii or microdiet to determine the effect of T. aceti on growth performance and survival rate of C. maraena. By analysing the fatty acid composition of T. aceti prior to and after enrichment with INVE spresso^® it was investigated whether the amount of n3‐polyunsaturated fatty acids (n3‐PUFA) in T. aceti could be further enhanced. Supplementation of Artemia nauplii with T. aceti increased growth significantly within the first 5 days of rearing in comparison to the non‐supplemented food treatments (14.39 ± 0.15 mm compared to 13.44 ± 0.18 mm; mean ± SE). However, growth and survival of juvenile C. maraena on nematode‐supplemented Artemia nauplii did not differ significantly from non‐supplemented Artemia nauplii at the end of the 14‐day rearing period (15.22 ± 0.15 mm compared to 14.86 ± 0.24 mm). All feeding treatments containing Artemia nauplii showed significantly higher growth and lower mortality at the end of the experiment in comparison to diets containing only the microdiet or T. aceti or a combination thereof. The overall low performance of T. aceti alone can most likely be explained by an insufficient capacity of C. maraena to digest this nematode species efficiently. Enrichment with INVE spresso^® successfully increased the proportion of DHA in the T. aceti tissue. The results reveal that T. aceti cannot be considered a full alternative to Artemia nauplii, at least not in the rearing of C. maraena, but might be a useful vector of essential fatty acids within the early rearing period of this and potentially other fish species when provided as live food along with Artemia nauplii.     

Monoclonal antibodies as reversible equilibrium carriers of radiopharmaceuticals

We have prepared monoclonal antibodies (MoAbs) with the specific ability to bind metal chelates such as ¹¹¹In benzyl EDTA. One, 10, 50 and 100 μg MoAb CHA255 K_b = 4 × 10E9 was complexed with ¹¹¹In BLEDTA II, BLEDTA IV, and benzyl EDTA and injected i.v. in Balb/c mice with KHJJ tumor. The biological half-life by whole body counting was profoundly altered for all three compounds; from minutes to hours with 10 μg; to days with 100 μg. Tumor uptake increased 50 fold at 24 h with increasing MoAb but satisfactory tumor concentrations (3% per g) and tumor/blood ratios (1.8:1) were obtained with an amount equivalent to 7 mg for a human. Blood level and whole body activity were decreased 30–50% within 3 h or i.v. injection of a “flushing” dose of unlabeled indium benzyl EDTA, increasing tumor/blood ratios to 50:1.     

Efficient Plant Genomic and Viral DNA Isolation from Mature Leaf Midrib of Banana (Musa spp)

The genomic DNA isolation from mature leaf midrib is a tough job, because of the abundance of polysaccharides and secondary metabolites, which interferes with DNA isolation as well as polymerase chain reaction (PCR) studies. The leaf midrib of 3^rd leaf from 3-moths old, ex-vitro developing banana [AAA, Dwarf Cavendish-Basrai (Sindhri banana)] plants (healthy and BBTV infected) was grinded in liquid N₂. Exact 0.3 g of leaf midrib powder was washed with washing buffer (100 mM Tris-Cl, 5 mM EDTA, 0.35 M sorbitol, 1% 2-mercaptoethanol) then homogenized in 0.8 ml of three different pre-heated (60°C) DNA isolation buffers. Supernatant was extracted through phenol: chloroform:isoamyl alcohol (25:24, v/v), chloroform: isoamyl alcohol (24:1, v/v) and finally with chloroform (100%) one by one. Maximum yields were ranged from 49.33 and 27.73 μg mg ^?1 DNA with impurities 5.67 and 5.87 μg mg^?1 through buffer I, while 45.77 and 25.53 μg mg^?1 DNA with 6.13 and 6.16 μg mg^?1 impurities through buffer III from healthy and infected plants respectively. Best one RAPD was observed in all the DNA samples isolated with different buffers, while viral amplification was good in DNA isolated with buffer I and II, when 10 (RAPD) and 25 ng DNA (C ₁ gene) was used as a template in a reaction of 25 μl. Meanwhile, buffer II is limited for viral DNA isolation while buffer I (1M Tris-Cl, 5M NaCl, 2 % cTAB, 50mM EDTA, 1 % PVP, 0.2 % 2-mercaptoethanol) has dual capacity for plant and virus DNA isolation. This described protocol is economic in terms of times, labor and cost.     

Ammonia toxicity as a criterion for the evaluation of larval quality in the prawn Macrobrachium rosenbergii

The feasibility of a short-term ammonia toxicity test as an evaluation criterion for larval quality was assessed in three trials. In each one, Macrobrachium rosenbergii larvae originating from the same spawn were nutritionally differentiated in two groups by feeding them either a nutrient-rich (Artemia nauplii enriched for 24 h with n-3 highly unsaturated fatty acids (HUFA) and ascorbic acid (AA)) or a nutrient-poor diet (Artemia nauplii starved for 24 h). Throughout their development, larvae from both treatments were exposed during 24 h to six concentrations of total ammonia (NH₄⁺+NH₃) and a control (no ammonia added). Based on mortality rates, the median lethal concentration for 50% of the population (LC₅₀) was estimated. As expected from earlier work, larvae fed the optimal diet presented higher n-3 HUFA and AA contents as well as higher growth and metamorphosis rates. From the moment the effect of diet quality was analytically detectable in the tissues of the larvae, the ammonia test was able to distinguish both groups of larvae. Differences in ammonia tolerance were observed as early as larval stage 4 and remained evident throughout larval development. The short-term ammonia toxicity test proved to be a valuable, sensitive and reproducible criterion for the establishment of larval quality.     

High-performance liquid chromatographic assay for methyl-β-cyclodextrin in plasma and cell lysate

This paper describes a high-performance liquid chromatographic method with fluorescence detection for the analysis of methyl-β-cyclodextrin (MEBCD) in plasma and cell lysate, after in situ complexation with 1-naphthol. The size-exclusion HPLC column packed with TSK 3000 SW gel, was equilibrated with an eluent mixture composed of methanol and purified water (2:98, v/v) containing 10⁻⁴ M 1-naphthol as a fluorophore. The detection is based on fluorescence enhancement caused by the formation of inclusion complexes and was performed at 290 and 360 nm for excitation and emission, respectively. The method involved a simple treatment of the samples with chloroform. Daunorubicin was used as internal standard. Limits of quantitation were 0.8 μM in plasma and 0.5 μM in cell lysate. Detection limits of 0.5 μM (50 pmol) and 0.3 μM (30 pmol) were obtained for MEBCD in the two media, respectively. Linear detection response was obtained for concentrations ranging from 1 to 100 μM in plasma and cell lysate. Recovery from plasma proved to be more than 40%. Precision, expressed as C.V. was in the range of 4 to 11%. Accuracy ranged from 89 to 105%.     

Rearing tench (Tinca tinca L.) larvae on live feed (Artemia) and on two transition schedules from live to dry diets

Two 60‐day experiments were carried out on tench (Tinca tinca L.) from day 5 post‐hatch. Density was 20 larvae L^?1 and temperature 24 ± 0.5°C. In experiment 1, Artemia nauplii were the sole food, testing nauplii amounts and feeding frequency. High survival rates (between 79.5% and 95.5%) were obtained. Growth was faster as nauplii amounts were greater; the highest growth rate (11.00), weight (265.5 mg) and Fulton’s coefficient (1.40) were obtained when fish were fed in excess once a day, without significant differences from the growth obtained by feeding in excess of eight times a day. In experiment 2, a dry diet for marine fish was tested as a replacement for Artemia nauplii, following two transition protocols, one faster than the other; high survival rates (between 77.7% and 87.1%) were again obtained. The slower transition allowed a growth rate of 10.14, length of 23.1 mm, weight of 158.3 mg and a Fulton’s coefficient of 1.28, without significant differences from the faster transition. At all stages, growth values were significantly higher from feeding nauplii in excess as the sole food, but the required nauplii quantity was six times higher than the amount supplied to the animals fed the dry diet.     

Food-web modification by an invertebrate predator in the Great Salt Lake (USA)

Summary During unusually wet years the salinity of the Great Salt Lake (Utah) decreased from above 100 g/L to 50 g/L. This allowed the predaceous insect Trichocorixa verticalis to invade the pelagic region of the lake and reach a mean summer density of 52/m³. Concurrent changes in the pelagic ecosystem were: a decrease in the dry biomass of the previously dominant filter-feeding brine shrimp Artemia franciscana from 720 to 2 mg/m³, the invasion of three other zooplankton taxa, a 10 × decrease in community filtration rate, a 20 × increase in chlorophyll a concentration, a 4 × decrease in water clarity and perhaps a decrease in soluble nutrients. Trichocorixa abundance was also inversely correlated with the abundance of Artemia along a salinity gradient in the lake's estuary. In a 9-d microcosm experiment Trichocorixa preyed on nauplii and decreased the total density of Artemia from 103 to 6/L. The reduction in Artemia allowed protozoans to increase 10–100 ×. Changes in chlorophyll and clarity were consistent with those observed in the lake. These results suggest that invertebrate predation may be an important factor structuring simple food webs such as those found in moderately saline lakes.